Fig. 7: ABL1 deficiency in C. elegans increases ATG-18 gene expression and autophagic flux.

a Hermaphrodites of the N2 wild type, abl-1(ok171) mutant, atg-18 (gk378) mutant or abl-1(ok171);atg-18 (gk378) double mutant strains were singly transferred to NGM plates, and eggs per hermaphrodite were counted over the whole reproductive period, n ≥ 4 (left panels). The number of L4 nematodes was calculated as a percentage of the total number of eggs laid (right panels). One-way ANOVA with Holm-Sidak post-hoc test, mean ± SD, n ≥ 4. b For the L1 starvation assay, eggs were isolated, and L1 larvae hatched in nutrient-free medium, in which they were kept for up to 34 days. Every 2-3 days, larvae were removed from starvation and spotted onto NGM/OP50 plates. After 2 days of unrestricted feeding, nematodes that reached the L2 larval stage or later were scored, and the percentage of developing larvae was calculated. The corresponding results for WT, abl-1(ok171) mutant, atg-18 (gk378) mutant or the abl-1(ok171);atg-18 (gk378) double mutant strain are shown. c N2 wild-type, and wild-type (GFP::LGG1) or abl-1(ok171) mutant (GFP::LGG1; abl-1(ok171) L1 larvae expressing the adIS2122 transgene GFP::LGG1 were starved for 16 h. Protein extracts from whole larvae were analysed by immunoblotting against GFP or tubulin (left panels). Relative protein levels of cleaved GFP over tubulin were quantified (right panel, Welch’s t test, mean ± SD, n = 3). d Likewise, wild-type (GFP::LGG1) or abl-1(ok171) mutant (GFP::LGG1; abl-1(ok171)) L1 larvae expressing the adIS2122 transgene GFP::LGG1 were imaged (left panels) and GFP-LGG1 puncta number (middle panels) as well as the mean puncta size (right panels) per nematode determined using CellProfiler. For statistical analysis, an unpaired t-test with Welch’s correction was performed (GFP::LGG1, 61 nematodes; GFP::LGG1; abl-1(ok171, 55 nematodes; mean ± SD). Scale bar = 50 µm. e Total RNA from synchronised and sterilised wild-type or abl-1(ok171) nematodes was extracted on day 1, day 6 and day 11 of adulthood. Relative ATG-18 mRNA levels were analysed (Welch’s t test, mean ± SD, n = 4). Left panel: ATG-18 expression in wild type nematodes. Middle panels: ATG-18 expression in abl-1(ok171) nematodes. Right panel: Additional comparative display of results (left panels, middle panels) of day 1 and day 11 only. Supplementary material is available (Supplementary Data 1). P values: *p < 0.05; **p < 0.01; ***p < 0.001; ns not significant.